This is a resubmission of the renewal application for my Research Project Grant (R01) entitled Regulation of Differentiation in Esophageal Epithelia (DK069984), which concluded on March 31, 2011. The overarching goal of this proposal is to understand the regulation of esophageal epithelial stratification, inflammation, and carcinogenesis through mechanistic studies of the key transcriptional regulator Kr|ppel-like factor 4 (KLF4). The significance of this proposal lies in (a) the prevalence of esophageal disorders, which are among the most common ailments in the United States and the world and (b) the important and emerging roles of KLF4 in the control of essential cellular processes in numerous tissues and cell types, including esophageal keratinocyctes. The PI is an experienced investigator who is an expert in the Kr|ppel-like factors (KLFs), animal models of disease, and esophageal squamous cell biology, and is supported by a superb research team, complemented by expert collaborators. In this proposal, we will take advantage of novel mouse models, which we have generated, and complementary in vitro systems utilizing two-dimensional and three-dimensional organotypic culture to test the hypothesis that KLF4 is a critical regulator within adult esophageal squamous epithelia, controlling processes such as keratinocyte migration and differentiation, the inflammatory response, and squamous cell cancer development. To explore the regulation of esophageal epithelial stratification, inflammation, and carcinogenesis by KLF4, we will undertake the following interrelated Specific Aims: (1) We will explore the function of KLF4 in esophageal epithelial migration and stratification (a) through mechanistic, epistatic studies of KLF4 and the KLF4 target WNT5A, a non-canonical Wnt, and (b) by identification of additional, relevant KLF4 target genes through functional genomics analyses~ (2) We will investigate the role of KLF4 loss in esophageal carcinogenesis by (a) examining the functional interaction of KLF4 and p53 in esophageal epithelial cells in vitro and in vivo, and (b) identifying the mechanisms of KLF4 loss in esophageal dysplasia and squamous cell cancer~ and (3) We will determine the mechanisms by which KLF4 promotes inflammation and the non-cell autonomous proliferative response by (a) evaluating the role of Rho Stases in NF:B activation by KLF4, (b) examining the effects of NF:B and TNF1 inhibition on proliferation of keratinocytes from ED-L2/KLF4 mice~ and (c) crossing ED-L2/KLF4 mice with esophageal-specific Ikk2 null mice. These complementary approaches will confirm and extend the findings from our current funding cycle, as outlined in Preliminary Data and our two recent publications in Gastroenterology. Moreover, the proposed research will be supported by the superb and collegial intellectual environment and the exceptional resources and facilities available to the PI and his team. We anticipate that these studies will provide insight into the factors that maintain normal esophageal epithelial homeostasis and the pathways that are disrupted in esophageal diseases, both benign and malignant.